The Mla system of diderm Firmicute Veillonella parvula reveals an ancestral transenvelope bridge for phospholipid trafficking

E. coli and most other diderm bacteria (those with two membranes) have an inner membrane enriched in glycerophospholipids (GPLs) and an asymmetric outer membrane (OM) containing GPLs in its inner leaflet and primarily lipopolysaccharides in its outer leaflet. In E. coli, this lipid asymmetry is maintained by the Mla system which consists of six proteins: the OM lipoprotein MlaA extracts GPLs from the outer leaflet, and the periplasmic chaperone MlaC transfers them across the periplasm to the inner membrane complex MlaBDEF. However, GPL trafficking still remains poorly understood, and has only been studied in a handful of model species. Here, we investigate GPL trafficking in Veillonella parvula, a diderm Firmicute with an Mla system that lacks MlaA and MlaC, but contains an elongated MlaD. V. parvula mla mutants display phenotypes characteristic of disrupted lipid asymmetry which can be suppressed by mutations in tamB, supporting that these two systems have opposite GPL trafficking functions across diverse bacterial lineages. Structural modelling and subcellular localisation assays suggest that V. parvula MlaD forms a transenvelope bridge, comprising a typical inner membrane-localised MCE domain and, in addition, an outer membrane ß-barrel. Phylogenomic analyses indicate that this elongated MlaD type is widely distributed across diderm bacteria and likely forms part of the ancestral functional core of the Mla system, which would be composed of MlaEFD only.

Comments 1. Ln 107-8: I don't think that it is accurate to claim there is a dogma that all OM biogenesis systems are the same as E. coli simple because other species have not been studied.This statement should be softened.2. The figures numbers are mislabeled in the figure legends.3. It is mentioned that an mlaE mutant was not obtained, but a triple mutant was produced.How often does mutant production fail in these species?Is it possible that deletion of mlaE in the presence the MlaDF is toxic?4. Ln 143: Was a homolog of pgsA identified? 5. Ln 145-6: This should be reworded to avoid confusion over whether all the phospholipid synthesis genes are encoded as part of a fused gene rather than just the plasmalogen genes.6. Fig. S3A: Reference lipids are listed as PC, PE, and CL in one TLC and the legend and as PG, PE, and CL in the other TLC.Is this accurate?Also, the text mentions whole cell and membrane extracts.It is unclear which are shown.7. Ln 196-8: Although I agree with the conclusions of the Mla system being involved in retrograde transport and TamB in anterograde transport, there must be at least one more anterograde phospholipid transporter if tamB can be deleted (unless the OM is not essential).Are there other AsmA-like proteins in V. parvula? 8. Fig. Ext 4A: It would be useful to have a view of the barrel from the top or bottom to be able to see whether the barrel has an open channel and how large the channel is. 9. Ln 281-3: This sentence is seems to imply that M. tuberculosis is monoderm.It is not gramnegative but does have twofold membranes (cytoplasmic membrane and mycomembrane).10.Fig. Ext 9: Are the disordered regions of the example MlaD homologs without barrels predicted to form a structure if modeled as a hexamer?
Reviewer #2 (Remarks to the Author): This study by Grasekamp et al. focuses on understanding the Mla system in Veillonella.The Mla system has been mainly studied in the "model" organism Escherichia coli.Mla is important for maintaining the asymmetric lipid structure of the outer membrane (OM) by transporting phospholipids from the outer leaflet of the OM to the inner membrane (IM) using several proteins including a soluble periplasmic shuttle protein, MlaC.Interestingly, Veillonella lacks MlaC, as well as the OM component MlaA, only sharing the core components of the IM Mla complex.The work presented here clearly shows that the Mla system in Veillonella functions like that of E. coli; however, it has a different structure.The authors show that one of the IM components, MlaD, is significantly larger in Veillonella than in E. coli, with a predicted long helical domain and C-terminal b-barrel.By generating AlphaFold and AlphaFold Multimer models and by monitoring protein localization in cells, the authors show that the large Veillonella MlaD bridges the IM and OM.Specifically, their structural models predict that an MlaD hexamer forms a tunnel-like helical structure that could cross the periplasm and connects to six beta-barrels predicted to be in the OM.The authors present phylogenetic studies and propose the evolution of the Mla system.They also point out that the "model" system from E. coli is more an exception than the rule.I enjoyed reading and thinking about this study.The experiments are well done, the data are clear, and the conclusions are justified.The manuscript is also well crafted.Overall, the work is impactful.It clearly demonstrates the great value of studying the cell envelope of "non-model" organisms, especially those that are distantly related to the traditional model organisms such as E. coli.Nice work!There are two main points for authors to consider: 1) What is the estimated length of the predicted tube-like structure that is proposed to cross the periplasm?Is the estimated length in agreement with the size of the periplasm in Veillonella cells?This information should be added to the manuscript.
2) OM vesicles: It would be important to include a control that rules out lysis, such as an immunoblot for a cytoplasmic protein.Also, how were samples standardized for the TLC shown in Fig. 1C?
Minor points: 3) Why is the mlaE single mutant not viable but the tripe mlaEFD viable?I assume that having MlaD and/or the ATPase MlaF without MlaE is lethal?It will be speculative (and should be stated as such), but it would be nice to include a possible explanation so that readers are not confused.
'" .:>6B%)&$%)(* H196 4?>CA2BC:>8 0/ @6A=623:<:CF @96>?CF@6B ?7 J=<2, 2>5 JC2=+# C?86C96A E:C9 C96 BCA:;:>8 4?=@<6=6>C2C:?>?7 C96 5?D3<6 J=<2,JC2=+ BCA2:>GI -C9:>; C96 DB6 ?7 "complementation" is incorrectly used here because deletions cannot complement anything.Instead, "suppression" or "cosuppression observed in the double mutant" should be used.5) Lines 202-203: "...understand how three IM proteins could be structurally arranged within the envelope of V. parvula to facilitate GPL trafficking in the absence of MlaABC."MlaA is a cytoplasmic protein that forms a complex with two IM proteins.In addition, the question does not really pertain to MlaB (cytoplasmic protein of unclear function).What is unclear is how the Mla system functions without the periplasmic shuttle/chaperone and OM component known to exist in E. coli.I find the text confusing.I suggest that the authors change it to something like "…understand how the predicted structure of the IM MlaEFD complex could facilitate GPL trafficking in V. parvula in the absence of the periplasmic and OM MlaCA components."6) Lines 220-221: "generating a model for the full length protein (Fig 4B)."Using what to generate the model?Information about using AlphaFold should be included here and in the legends of Ext.Fig 3 and 4. Also, it should be "full-length protein".7) Lines 375-378: Reference 40 only showed the structure of a section of TamB.The authors might want to consider referencing recent publications that include AlphaFold predicted structures for TamB and other AsmA-like proteins.For example: PMIDs 37455811, 36571082, and 34781743.
8) The authors should clearly state that the structures presented are models or predicted structures throughout the manuscript.I recommend adding "predicted" or "model" in front of "structure(s)" when referring to models.This is especially important in the Discussion when discussing the MlaD proteins that are large but not predicted to have a beta-barrel.I doubt that the C-termini of those proteins look like spaghetti.Either the model is wrong, or a partner(s) is missing that completes the fold.9) Check labels in Fig. S3 for i and ii panels.Labels have been cut off or are missing.
Reviewer #3 (Remarks to the Author): Grasekamp, et al. investigate the possible role of an MCE transport system in the transport of glycerophospholipid (GPL) for the maintenance of the outer membrane in the non-model diderm species, Veillonella parvula.Based upon distant similarity to some of the components of the Mla system in E. coli, the authors refer to the V. parvula system as "Mla" and hypothesize that it may have a similar function in maintaining the asymmetry of the outer membrane; indeed, the authors show that mutations in the V. parvula system result in similar phenotypes, such as detergent hypersensitivity, increased vancomycin resistance, and hypervesiculation.The authors also show that loss of tamB in a mlaD KO background partially rescues the detergent hypersensitivity phenotype, suggesting that MlaEFD and TamB play opposing roles (retrograde and anterograde trafficking, respectively) in maintaining GPL homeostasis of the V. parvula outer membrane (OM).However, there are substantial differences between E. coli Mla and the V. parvula system, which makes this work quite interesting.Structural modeling and subcellular localisation experiments suggest that the V. parvula MCE protein may form a transenvelope tunnel that is anchored in both the IM (inner membrane) and OM.Intriguingly, the authors use bioinformatics to show that MCE proteins like the one from V. parvula are widespread across diderm bacteria, and may perhaps be similar to the most ancestral form of the transporter.
Overall, we feel like this is a very interesting paper that adds to our understanding of this diverse family of transporters.The results are generally clear, experiments appear to be well executed, and the use of AlphaFold was appropriate, thoughtfully analyzed, and presented with the right degree of "skepticism" (it is super powerful, but sometimes does funny things, like it did here in the helical tunnel vs groove predictions).The paper was posted as a pre-print on BioRxiv, is wellwritten, and was a pleasure to read.We don't have any very serious concerns, but have a number of suggestions for improving the final version.

MAJOR COMMENTS:
None MINOR COMMENTS: We would suggest referring to the proposed complex as forming a "tunnel" instead of a "bridge", since bridges usually leave the cargo open to the environment (like the LPS exporter bridge), while tunnels completely surround the cargo (like in an efflux pump).
We think the authors should call the V. parvula system something other than "Mla".There is certainly some similarity to E. coli Mla, but there are also substantial differences (as the authors point out, only 3 of the 7 proteins found in E. coli Mla have counterparts in the V. parvula system).It is also worth noting that there are bacterial species like P. aeruginosa that have a bonafide Mla system as well as a V. parvula-like MCE system, which would make calling both "Mla" a bit awkward.I think the best characterized V. parvula-like MCE system is called TGD in A. thaliana; perhaps a new name could be based on that, or could be something completely new of the authors choosing.I think this will help avoid confusion in the field.
The authors show that the V. parvula mla KO strains exhibit increased resistance to vancomycin, but this phenotype does not appear to be complemented by adding back in the deleted genes.Was a similar pattern observed for E. coli mla mutants?Why can't this be complemented?SDS/EDTA sensitivity assay: The concentrations of SDS reported (~0.003-0.004%)are ~100-fold lower than what is typically used for E. coli mla mutants.We would like to confirm that these values are correct and not a typo.
Have the authors tried predicting a homohexamer of the beta-barrel domain?If they are analogous to MlaA, they would need to create a pathway for lipids to move from within the OM to the tunnel through the MCE protein.It would be interesting if AF suggests they form a stable assembly, vs each barrel doing its own thing in isolation.
ln 71: Perhaps it would be appropriate to ref the earlier work establishing the IM complex here: Kamischke 2019, Ekiert 2017, and Thong 2016; if there is space, one could also cite the more recent 6 papers for the high resolution structures that all came out around the same time (PMIDs: 33845086, 34188171, 33298869, 33199922, 32884137, 33236984).In the context of the MlaA-Omp complex, we could also imagine citing Abellon-Ruiz 2017 here.
Fig. 1 legend: The authors refer to the MCE protein as a "substrate binding protein", akin to MBP in a classical ABC importer.However, the MCE protein is playing a completely different role (if anything, MlaC would be analogous to a SBP, but that is absent in the V. parvula system).We suggest removing this terminology.
Line 118-121: The authors mention making single mlaDEF mutations and that all V. parvula mla mutants display hypersensitivity to SDS/EDTA and increased resistance to vancomycin.However, not all of this data is shown, so this claim is not well supported.We suggest showing all the data or altering the claim to match the data shown.ln 131: "these phenotypes are indicative of a loss of lipid asymmetry": We might suggest softening the claim here.This may be correct, but OM asymmetry has not been directly assessed, and this system appears to be quite different from mla.It may be remodeling the OM in a different way, and we think the observed phenotypes don't necessarily have to arise from a loss of asymmetry.ln 151-153: Is there any precedent for vinyl ether bonds in bacterial lipids?Perhaps a reference to prior work here would be appropriate, and a statement of how common/uncommon these are?ln 176-177: Are these 2 independent insertions at the exact same location?ln 179-180: How many AsmA-like proteins are encoded in the genome?This may affect the claim made in ln 196-198, which could perhaps be softened a bit to avoid being overly speculative.ln 203-204: Does that mean that MlaF has a long C-terminal tail that may "handshake" with the neighboring MlaF subunit, even in the absence of MlaB?Or is the V. parvula MlaF a little shorter?Also, in other MlaE structures there is an N-terminal amphipathic helix that runs parallel to the plane of the membrane, in the cytoplasmic leaflet.I can see it in Ext.Data Fig. 3D for E. coli but can't pick it out for the V. parvula prediction.Is it missing in V. parvula MlaE?I think it would be worth 1 sentence commenting on these differences if they are indeed different.ln 281-283: We think mycobacteria are regarded by many in the field as diderm (though I have had heated debates about this with colleagues), while we agree that many other actinobacteria are monoderms.Cryo ET of mycobacteria reveals an OM that appears very much like the OM of Gramnegatives (e.g., Hoffman, PNAS, 2008).So we suggest removing the specific mention of M. tuberculosis as a monoderm.Are the strips for each strain from the same plate, or is this a composite image?In order to compare the suppressor mutants to the controls, they need to be on the same plate, but I'm not sure that this is the case?Fig. 3C: Please indicate in the legend what the dashed line box is highlighting (i.e. the hydrophobic regions that are predicted to be membrane-embedded).Also, perhaps this is due to the angle of the model, are there only three C-terminal beta barrels (reflecting the possibility that several betabarrels can fuse to form a trimeric conformation, as mentioned in the Discussion) or are another three just hidden from view?Fig. 5: We think it would be very helpful to have three different categories of "MlaD" proteins, and color code them differently (currently, they are all one shade): 1) true MlaD proteins (with short Cterminal helical region); 2) Single MCE domain proteins with a long helical region, with betabarrels; and 3) Single MCE domain proteins with a long helical region, WITHOUT beta-barrels.

Main Text Figures: The middle
Extended Fig. 3A: Label the figure with "i" and "ii" as written in the legend.Also, the legend mentions that the major lipid species in V. parvula are labeled 1-4 in the figure, however the #4 in Aii looks like it runs similarly to the cardiolipin standard.This is a little confusing as the text mentions that there is no cardiolipin detected by MALDI-QIF-TOF.
Extended Fig. 3Bi: The sentence "The relative decrease in these two species from the mla KO OMVs confirmed the relative enrichment of PE" is a little confusing.Is there a typo here or can the authors elaborate more on the connection between the reduction of unknown lipid and PE increase.Extended Fig. 6: "Presence OF absence of Mla components…" I believe should be "Presence OR absence".We also cannot read the detailed species names, as well as for Ext.Fig. 7.
In order to make the AlphaFold predictions readily accessible to the community, coordinates should either be deposited in ModelArchive or included as supplementary files as part of the publication.

Reviewer #1 (Remarks to the Author):
In this work "Bridges instead of boats?The Mla system of diderm Firmicute Veillonella parvula reveals an ancestral transenvelope core of phospholipid trafficking."),Grasekamp, et al. investigate inter membrane phospholipid trafficking in the diderm Firmicute, Veillonella parvula.The authors identify a system with homology to part of the E. coli Mla system and determine that this system functions in retrograde phospholipid transport using a somewhat different mechanism than E. coli, mediated by a trans envelope architecture of MlaD.Further, the authors identify suppressor mutants in TamB suggesting that at least some anterograde transport is mediated by TamB.This paper, which is well-written, should be of interest to a very broad audience as it brings together biochemical, genetic, computational, and evolutionary approaches to investigate intermembrane phospholipid trafficking in a non-model organism and to investigate the application of the finding across bacteria.The findings are significant and the approaches are rigorous.However, there are several points that need clarification.
Comments 1. Ln 107-8: I don't think that it is accurate to claim there is a dogma that all OM biogenesis systems are the same as E. coli simple because other species have not been studied.This statement should be softened.

Answer:
We have softened this statement by replacing this sentence by: "Together, our results uncover novel functional information about GPL trafficking in a non-model organism, shedding light on the evolution of the Mla system, challenging the assumption that OM biogenesis systems studied in E. coli represent the majority of diderm bacteria, and highlighting the diversity in the OM biogenesis and maintenance systems." 2. The figures numbers are mislabeled in the figure legends.Answer: Thank you, this has been corrected.
3. It is mentioned that an mlaE mutant was not obtained, but a triple mutant was produced.How often does mutant production fail in these species?Is it possible that deletion of mlaE in the presence the MlaDF is toxic?Answer: Although V. parvula is not always the easiest microorganism to manipulate, construction of mutants rarely fails in this species unless we encounter a problem of toxicity or essentiality.We favour the same hypothesis as one of the other reviewers to explain our incapacity to construct the mlaE mutant, and have added this hypothesis at lines 113-114: "-VISH SHE EWCEOSINM NF SHE ZmlaE mutant that could not be obtained, perhaps due to a toxicity of MlaD and/or MlaF in the absence of MlaE -«. 4. Ln 143: Was a homolog of pgsA identified?Answer: As described in supplementary Fig. 1A (now supplementary Fig. 3), several homologues of genes involved in glycerophospholipid biosynthesis have been identified in V. parvula.FNLLGLLA_00889 encodes a protein that displays homology to both Pss and PgsA, though further work is required to determine its function.This has been corrected in the now Supplementary Fig. 3A.7. Ln 196-8: Although I agree with the conclusions of the Mla system being involved in retrograde transport and TamB in anterograde transport, there must be at least one more anterograde phospholipid transporter if tamB can be deleted (unless the OM is not essential).Are there other AsmA-like proteins in V. parvula?Answer: Yes, we did identify one other gene encoding a potential AsmA-like protein in Vp.We constructed all combinations of mutants of mlaD, tamB and "asmA".AsmA does not appear to have a significant impact on ;9 HNLENRSARIR IM SHE CNLBIMASINMR NF DEKESINM SHAS VEQE SERSED' 7MDEED% EUEM SHE SQIOKE ZmlaDZtamBZasmA mutant is viable, suggesting there are other unidentified PL trafficking systems in V. parvula.We also see that deletion of asmA in the mlaD background does not suppress the associated OM permeability phenotype, nor that of the hypervesiculation.
Considering that this gene was also not identified in any suppressor or transposon mutagenesis screens for the mlaD mutant, we decided not to introduce supplementary information on it to simplify our message.However, we will likely use this information for a future study as we continue to probe the systems which govern PL homeostasis in V.parvula.
We have added the information of the existence this AsmA-like protein in Vp at the end of the discussion section and indicate that further work will be necessary to clarify which function are exactly performed by TamB and this AsmA-like protein and to identify potential other genes involved in PLs transport in Vp: Lines 363-363: "Indeed, one other asmA-like protein exists in V. parvula, and further work is required to elucidate its functional role, if any, in the context of OM homeostasis."8. Fig. Ext 4A: It would be useful to have a view of the barrel from the top or bottom to be able to see whether the barrel has an open channel and how large the channel is.

Answer:
As the pore channel is quite narrow and not straight, a top or bottom view does not allow to see the channel.Instead, we now present in Supplementary Fig. 9C (formerly Extended Fig4C) a representation of the channel as computed by the HOLE software.9. Ln 281-3: This sentence seems to imply that M. tuberculosis is monoderm.It is not gram-negative but does have twofold membranes (cytoplasmic membrane and mycomembrane).

Answer:
Thank you for this comment.Yes the mycomembrane is clearly an outer membrane, but emerged "de novo" in the clade to which M. tuberculosis belongs.We were referring to the phylum as being ancestrally monoderm.We agree that this can be confusing and have removed the sentence for simplicity.14) (526226_Gbronchialis@ACY20515.1 and 83332_Mtuberculosis@CCP43333.1).In both cases, the confidence scores returned by AlphaFold (plDDT, pTM and ipTM) were very low and no additional folding was observed.Of note, 83332_Mtuberculosis@CCP43333.1 has 65% identity with M. smegmatis mce1f protein from the Mce1 transporter for which the structure was reported recently (Chen et al., Nature.2023 Aug;620(7973):445-452.doi: 10.1038/s41586-023-06366-0) and in which the C-ter of the 6 different Mce1 proteins form a small barrel.We thus cannot exclude that some of the "without barrel" MlaD homologues analysed here do not self-associate to form a hexamer but rather form a complex with other MlaD-related proteins.However, our AlphaFold modelling as hexamer of 16 more "without barrel" MlaD sequences revealed that two of them possibly form tiny barrels, with a hydrophobic core.This further suggests a potential convergent evolution leading to homo-or heteromeric ß-barrels.This information is now provided in a new Supplementary Figure 15 (see below) and further discussed in lines 307-311.This study by Grasekamp et al. focuses on understanding the Mla system in Veillonella.The Mla system has been mainly studied in the "model" organism Escherichia coli.Mla is important for maintaining the asymmetric lipid structure of the outer membrane (OM) by transporting phospholipids from the outer leaflet of the OM to the inner membrane (IM) using several proteins including a soluble periplasmic shuttle protein, MlaC.Interestingly, Veillonella lacks MlaC, as well as the OM component MlaA, only sharing the core components of the IM Mla complex.The work presented here clearly shows that the Mla system in Veillonella functions like that of E. coli; however, it has a different structure.The authors show that one of the IM components, MlaD, is significantly larger in Veillonella than in E. coli, with a predicted long helical domain and C-terminal b-barrel.By generating AlphaFold and AlphaFold Multimer models and by monitoring protein localization in cells, the authors show that the large Veillonella MlaD bridges the IM and OM.Specifically, their structural models predict that an MlaD hexamer forms a tunnel-like helical structure that could cross the periplasm and connects to six beta-barrels predicted to be in the OM.The authors present phylogenetic studies and propose the evolution of the Mla system.They also point out that the "model" system from E. coli is more an exception than the rule.
I enjoyed reading and thinking about this study.The experiments are well done, the data are clear, and the conclusions are justified.The manuscript is also well crafted.Overall, the work is impactful.It clearly demonstrates the great value of studying the cell envelope of "non-model" organisms, especially those that are distantly related to the traditional model organisms such as E. coli.Nice work!There are two main points for authors to consider: 1) What is the estimated length of the predicted tube-like structure that is proposed to cross the periplasm?Is the estimated length in agreement with the size of the periplasm in Veillonella cells?This information should be added to the manuscript.

Answer:
From the top of the TM-helix to the bottom of the OM beta-barrel ring, the length is 18.5 nm, slightly lower than the commonly reported periplasmic width of 21 nm in E coli doi.org/10.1128/jb.185.20.6112-6118.2003).However, the beta-hairpin connecting the last central helix and the beta-barrel (displaying low plDDT values) could adopt a more extended conformation, adding up 5 nm more, for a total possible length of 23.5 nm.An accurate measurement of Vp periplasm size using cryo-EM images leads to a width of 24.2 ± 1.8 nm (n=26), so the maximum length of the tube-like structure is compatible with our estimation of the Vp periplasm width.This has been added to the manuscript (lines 231-233): "8IJE SHERE QERNKUED RSQTCSTQER% SHE OQEDICSED STMMEK FNQLED BX SHE ^&HEKICER GEMEQASER A HXDQNOHNBIC IMSEQINQ of ~13 Å in diameter (Fig 4C ) -supporting the possibility of a role in hydrophobic substrate transport -and a total possible length of ~23.5nm, compatible with the periplasmic width in V. parvula (24.2 ±1.8 nm (n=26) as measured by cryo-EM." 2) OM vesicles: It would be important to include a control that rules out lysis, such as an immunoblot for a cytoplasmic protein.Also, how were samples standardized for the TLC shown in Fig. 1C .$%VHICH IR AKRN RTOONQSED BX KISSKE DIFFEQEMCE IM <:2REP OQNFIKER BESVEEM @> AMD Zmla strains, even under conditions of slight stress (data not shown).More importantly, however, our NanoFCM data generated from both whole-cell cultures, and of extracted and purified OMV samples, display the same average scattering profiles for 'small events' or vesicles, with an average diameter of ~60 nm (as shown in Supplementary Fig 7), suggesting that these extracted samples are indeed vesicles and not random fragments of cell membrane that recombine.This is further supported by the average 60 nm diameter of OMVs observed in tomograms GEMEQASED FQNL ZmlaD cultures.This information is now provided in the Methods section: Lines 615-616: "Purified OMVs were analysed via NanoFCM confirming an average size of ~60 nm, matching the average size of vesicles in whole cell cultures observed via both NanoFCM and cryo-electron tomography (Supplementary Fig 7).These observations of OMVs preparation are compatible with absence of cell lysis as also observed in cfu CNTMSIMG EWOEQILEMSR NF @> AMD ZmlaD strains (see Fig 2,Supplementary Figs 2 & 6)." To rule out the presence of lysis, we did indeed attempt immunoblotting against OMV samples with antibodies against both TolC and SecA, as previously used for the membrane fraction localisation.Expectedly, no signal was generated for SecA, the inner membrane control.However, no signal was found for TolC, the OM control, either.We posit that TolC may be excluded from vesicle formation as it is connected to inner membrane proteins.This would not prevent it from being used as an OM marker for fractionation because of the French press lysis process, but makes it a very inconvenient marker for OMVs.We are trying to obtain a new antibody targeting the OM for our OmpM related studies, which may yield better results, but this is out of scope for this paper.Another ongoing development is the use of the enzymatic activity of catalase and NADH oxidase as a reporter for the presence of cytoplasmic and periplasmic membrane contamination in OMVs or in membrane fractionation assays.
For the standardisation of TLCs, lipids were extracted from the same volume of cell cultures normalised by OD600 (or in the case of OMVs, the same volume of supernatant) and resultant extracted lipid pellets were weighed and resuspended in the corresponding volume of chloroform before loading the same volume of each extracted lipid sample (10bl) onto the silica plates.However, taking into account both the variabilities in lipid extraction and weighing small quantities of lipids, we think it is more cautious to refer to the relative enrichment of PE within a sample as compared to other lipid species -such as the ratio of PE to lipid 2 -rather than quantities.
We have added the information in the Methods section and rephrased the manuscript accordingly: Lines 158-160: "2CQNRR AKK INDIME UAONTQ&RSAIMED OKASER% ;9?R OQNDTCED BX ZmlaD strains display a relative enrichment for PE when compared to another lipid species that is abundant in WT OMVs (Fig 2c

, Supplementary Fig 5b)."
As we continue to develop our toolkit for lipid analyses in V. parvula, we will generate more quantitative data to further interrogate the functioning of these genes in OM homeostasis and maintenance.
Minor points: 3) Why is the mlaE single mutant not viable but the triple mlaEFD viable?I assume that having MlaD and/or the ATPase MlaF without MlaE is lethal?It will be speculative (and should be stated as such), but it would be nice to include a possible explanation so that readers are not confused.

Answer:
Thank you for pointing to this aspect.As stated in our answer to reviewer 1, we favour the hypothesis that having MlaD and/or the ATPase MlaF without MlaE is toxic for the cell.
We have added this hypothesis in lines 113-114: "-VISH SHE EWCEOSINM NF SHE ZmlaE mutant that could not be obtained, perhaps due to a toxicity of MlaD and/or MlaF in the absence of MlaE -«.

Answer:
Thank you for this comment -we have modified this sentence for clarity: ">HE CNMSQARSIMG ;9 OEQLEABIKISX OHEMNSXOER NF ZmlaD AMD ZtamB, together with the striking suppression of SHE ZmlaD OM related phenotype by the tamB mutation, hints to the antagonistic functions of these two genes in envelope biogenesis and maintenance."5) Lines 202-203: "...understand how three IM proteins could be structurally arranged within the envelope of V. parvula to facilitate GPL trafficking in the absence of MlaABC."MlaA is a cytoplasmic protein that forms a complex with two IM proteins.In addition, the question does not really pertain to MlaB (cytoplasmic protein of unclear function).What is unclear is how the Mla system functions without the periplasmic shuttle/chaperone and OM component known to exist in E. coli.I find the text confusing.I suggest that the authors change it to something like "…understand how the predicted structure of the IM MlaEFD complex could facilitate GPL trafficking in V. parvula in the absence of the periplasmic and OM MlaCA components."Answer: Thank you for this proposition.We have modified the sentence as suggested.
6) Lines 220-221: "generating a model for the full length protein (Fig 4B)."Using what to generate the model?Information about using AlphaFold should be included here and in the legends of Ext.Fig 3 and 4. Also, it should be "full-length protein".

Answer:
We have now modified the sentence from: "Considering all characterised MCE proteins to date form hexamers, we merged overlapping predicted structures of all domains of MlaDVp with hexameric stoichiometry, generating a model for the full-length protein (Fig 4b)."To: "Considering all characterised MCE proteins to date form hexamers, we built a structural model of the fulllength protein by merging models obtained with AlphaFold-Multimer 29,30 of hexameric configuration of all domains of MlaDVp with overlapping segments (Fig 4b).
We also have modified the legend of the corresponding figures accordingly.7) Lines 375-378: Reference 40 only showed the structure of a section of TamB.The authors might want to consider referencing recent publications that include AlphaFold predicted structures for TamB and other AsmA-like proteins.For example: PMIDs 37455811, 36571082, and 34781743.

Answer:
Thank you -we have now added the corresponding publications.
8) The authors should clearly state that the structures presented are models or predicted structures throughout the manuscript.I recommend adding "predicted" or "model" in front of "structure(s)" when referring to models.This is especially important in the Discussion when discussing the MlaD proteins that are large but not predicted to have a beta-barrel.I doubt that the C-termini of those proteins look like spaghetti.Either the model is wrong, or a partner(s) is missing that completes the fold.

Answer:
Thank you for your comment; we have added "predicted" where necessary.We agree with the reviewer that there is likely much to discover regarding this C-terminal region of long "barrel-less" MlaD, and we elaborated on this already in the discussion section, as well as the possibility of accessory proteins required for the proper folding / functioning of these transenvelope proteins.9) Check labels in Fig. S3 for i and ii panels.Labels have been cut off or are missing.Answer: Thank you, this has been modified.

Reviewer #3 (Remarks to the Author):
Grasekamp, et al. investigate the possible role of an MCE transport system in the transport of glycerophospholipid (GPL) for the maintenance of the outer membrane in the non-model diderm species, Veillonella parvula.Based upon distant similarity to some of the components of the Mla system in E. coli, the authors refer to the V. parvula system as "Mla" and hypothesize that it may have a similar function in maintaining the asymmetry of the outer membrane; indeed, the authors show that mutations in the V. parvula system result in similar phenotypes, such as detergent hypersensitivity, increased vancomycin resistance, and hypervesiculation.The authors also show that loss of tamB in a mlaD KO background partially rescues the detergent hypersensitivity phenotype, suggesting that MlaEFD and TamB play opposing roles (retrograde and anterograde trafficking, respectively) in maintaining GPL homeostasis of the V. parvula outer membrane (OM).However, there are substantial differences between E. coli Mla and the V. parvula system, which makes this work quite interesting.Structural modeling and subcellular localisation experiments suggest that the V. parvula MCE protein may form a transenvelope tunnel that is anchored in both the IM (inner membrane) and OM.Intriguingly, the authors use bioinformatics to show that MCE proteins like the one from V. parvula are widespread across diderm bacteria, and may perhaps be similar to the most ancestral form of the transporter.
Overall, we feel like this is a very interesting paper that adds to our understanding of this diverse family of transporters.The results are generally clear, experiments appear to be well executed, and the use of AlphaFold was appropriate, thoughtfully analyzed, and presented with the right degree of "skepticism" (it is super powerful, but sometimes does funny things, like it did here in the helical tunnel vs groove predictions).The paper was posted as a pre-print on BioRxiv, is well-written, and was a pleasure to read.We don't have any very serious concerns, but have a number of suggestions for improving the final version.

MAJOR COMMENTS:
None MINOR COMMENTS: -We would suggest referring to the proposed complex as forming a "tunnel" instead of a "bridge", since bridges usually leave the cargo open to the environment (like the LPS exporter bridge), while tunnels completely surround the cargo (like in an efflux pump).Answer: Thank you for this suggestion, and we agree with the reviewer on the formal definition of bridge vs tunnel.The term bridge that we used in the title, and to describe what we think could be a transenvelope system, is used here more as a generic term.As indicated by the reviewer, predicted structures by Alphafold are models and we cannot today be sure that the real structure taken by Vp MlaD is actually a closed tunnel.The use of the term tunnel in the title could therefore be misleading, while using a generic term bridge (e.g. a connecting structure between the two membranes) seems more appropriate at this stage of our knowledge.
-We think the authors should call the V. parvula system something other than "Mla".There is certainly some similarity to E. coli Mla, but there are also substantial differences (as the authors point out, only 3 of the 7 proteins found in E. coli Mla have counterparts in the V. parvula system).It is also worth noting that there are bacterial species like P. aeruginosa that have a bonafide Mla system as well as a V. parvula-like MCE system, which would make calling both "Mla" a bit awkward.I think the best characterized V. parvula-like MCE system is called TGD in A. thaliana; perhaps a new name could be based on that, or could be something completely new of the authors choosing.I think this will help avoid confusion in the field.

Answer:
Although we understand this comment, we feel renaming these proteins -which are homologous to characterized Mla proteins and still perform Mla (maintenance of lipid asymmetry) functions -would bring about more confusion in the field.Considering MlaEFD are the initially occurring proteins in evolution, and that this system has adapted and become more complex with the addition of MlaABC in the Proteobacteria, we are comfortable in maintaining MlaEFD as descriptors.This is also necessary considering the high homology between MlaE, MlaF, and the first 'half' of MlaD, in our new model and in other classical diderms.
-The authors show that the V. parvula mla KO strains exhibit increased resistance to vancomycin, but this phenotype does not appear to be complemented by adding back in the deleted genes.Was a similar pattern observed for E. coli mla mutants?Why can't this be complemented?Answer: We agree that this lack of complementation is unusual, and do not yet fully understand this phenomenon.Interestingly, vancomycin resistance of mla-deficient strains has been described in several other bacterial strains -including E. coli, A. baumannii and N. gonorrhoeae -and is so widely accepted that results published by Kamischke et al. were refuted by reviewers, as the authors initially reported an increased sensitivity of A. baumannii Zmla mutants to vancomycin (Kamischke et al., 2019).Further, the increased UAMCNLXCIM QERIRSAMCE NF ZmlaA mutants of N. gonorrhoeae were also unable to be complemented by reintroducing mlaA into the mutant (Baarda et al., 2019).
We propose that deeper, global envelope changes are triggered by the deletion of these mla genes, possibly involving a complex network of other pathways that cannot be restored upon reintroduction of Mla on a plasmid vector (notably due to problems in restoring the exact stoichiometry of the Mla proteins).Considering the robust complementation of the SDS / EDTA sensitivity phenotype, we expect the vancomycin resistance phenotype is more complex, and may be informed by RNAseq studies.
Interestingly, although vancomycin resistance of mla deletion mutants is widely accepted in the field, we do not find evidence of published complementation for this phenotype -nor, therefore, of a lack of complementation either.We wanted to be fully transparent and share this phenotype, and its lack of complementation, in case this has implications for our global understanding of the Mla system in the future.
-SDS/EDTA sensitivity assay: The concentrations of SDS reported (~0.003-0.004%)are ~100-fold lower than what is typically used for E. coli mla mutants.We would like to confirm that these values are correct and not a typo.

Answer:
Thank you for checking -yes, these are not typos.Vp is much more sensitive to SDS than E. coli, which may reflect a difference in composition of its outer membrane.We also expect this difference in OM permeability may reflect the difference in niches between these two organisms; whilst Vp typically inhabits the oral tract, E. coli must adapt to the gastrointestinal tract by resisting the action of host detergents, i.e. bile salts.
-Have the authors tried predicting a homohexamer of the beta-barrel domain?If they are analogous to MlaA, they would need to create a pathway for lipids to move from within the OM to the tunnel through the MCE protein.It would be interesting if AF suggests they form a stable assembly, vs each barrel doing its own thing in isolation.

Answer:
We indeed tried to model homo-hexamers of the C-terminal beta-barrel domain alone.While AlphaFoldmultimer predicts a tighter ring of six beta-barrels, the prediction confidence scores are very low (pTM of 0.279 and ipTM of 0.144 for the highest-ranked model); and the PAE is close to the maximum error (30 $) between beta-barrels, so we do not consider this arrangement as a reliable prediction.This information is now provided in Supplementary Fig 16 below.-ln 71: Perhaps it would be appropriate to ref the earlier work establishing the IM complex here: Kamischke 2019, Ekiert 2017, and Thong 2016; if there is space, one could also cite the more recent 6 papers for the high resolution structures that all came out around the same time (PMIDs: 33845086, 34188171, 33298869, 33199922, 32884137, 33236984).In the context of the MlaA-Omp complex, we could also imagine citing Abellon-Ruiz 2017 here.Answer: Thank you, we have added some of the corresponding references.As the introduction initially stated (now line 62) "In E. coli, the Mla system is composed of…" we had only included structures / papers within this organism.However, considering how crucial these articles are (which we do mention later in our manuscript) we have adapted this sentence to: "In model diderms such as E. coli and A. baumannii, the Mla system is composed of…" and added the suggested references.
-Fig. 1 legend: The authors refer to the MCE protein as a "substrate binding protein", akin to MBP in a classical ABC importer.However, the MCE protein is playing a completely different role (if anything, MlaC would be analogous to a SBP, but that is absent in the V. parvula system).We suggest removing this terminology.

Answer:
Thank you, we have removed this terminology.
-Line 118-121: The authors mention making single mlaDEF mutations and that all V. parvula mla mutants display hypersensitivity to SDS/EDTA and increased resistance to vancomycin.However, not all of this data is shown, so this claim is not well supported.We suggest showing all the data or altering the claim to match the data shown.-ln 131: "these phenotypes are indicative of a loss of lipid asymmetry": We might suggest softening the claim here.This may be correct, but OM asymmetry has not been directly assessed, and this system appears to be quite different from mla.It may be remodeling the OM in a different way, and we think the observed phenotypes don't necessarily have to arise from a loss of asymmetry.

Answer:
We have softened the claim by modifying the sentence to: "Together, these phenotypes are indicative of a remodeling of the OM and possibly a loss of lipid asymmetry".
-ln 151-153: Is there any precedent for vinyl ether bonds in bacterial lipids?Perhaps a reference to prior work here would be appropriate, and a statement of how common/uncommon these are?Answer: Yes, plasmalogens (a type of ether lipid, which are lipids containing vinyl ether bonds as opposed to ester bonds) are typically restricted to anaerobic bacterial species. 2 references have been added.We have also added clarity that the presence of vinyl ether bonds is indicative of plasmalogens / ether lipids.Lines 146-149: "NMR analyses also revealed that ~15-35% of each lipid species present in the envelope of V. parvula contains a vinyl ether bond (indicative of plasmalogens 27,28 ) which could result from the activity of the identified plasmalogen biosynthesis homologue".
3 Figures need to be re-labeled to match the in-text references (the second Fig.1" Fig.2, the current Fig.2" Fig.3and the current Fig.3" Fig.4).

Fig 3A:
Fig 3A:Are the strips for each strain from the same plate, or is this a composite image?In order to compare the suppressor mutants to the controls, they need to be on the same plate, but I'm not sure that this is the case?

Extended Fig. 5 :
In the legend, A, B, D, E refer to hexameric chains A to G. Should this not be A to F, as labeled in the figure?It would be more useful in understanding the figure if the legend detailed what each dashed line box was highlighting (around rank 4 of A, B, C and the AlphaFold models of C and F. Also, having the PAE colored error bar labeled with "high error" and "low error" as in Ext.Fig.3Cwould be helpful (can only label one representative error bar so as to not clutter the figure).
5. Ln 145-6: This should be reworded to avoid confusion over whether all the phospholipid synthesis genes are encoded as part of a fused gene rather than just the plasmalogen genes.Answer:Thank you -we have split the sentence to avoid confusion.Only the plasmalogen gene appears to encode the function of both PlsA and PlsR.6. Fig. S3A: Reference lipids are listed as PC, PE, and CL in one TLC and the legend and as PG, PE, and CL in the other TLC.Is this accurate?Answer: Thank you for pointing out this typing mistake.This has been corrected (PC for both TLCs).Also, the text mentions whole cell and membrane extracts.It is unclear which are shown.Answer: Thank you, only whole-cell extracts are shown for the first panel (A) and the secondary panels show OMVderived extracts -we have now added details of this in the figure legend and title in Supp Fig 3 (now renamed Supp Fig 5).
10. Fig. Ext 9: Are the disordered regions of the example MlaD homologs without barrels predicted to form a structure if modeled as a hexamer?Answer: Thank you for this interesting remark.Using AlphaFold2-Multimer, we predicted the structure of hexamers for the C-terminal domain (including the last segment of the helical stalk) of the 2 examples MlaD homologues shown in Extended Fig 9 (now Supplementary Fig.